Fig 1.
A simplified schematic representing the agrocinopine A and agrocin 84 roles in A. tumefaciens C58.
Upon infection, virulent agrobacteria transfer a small DNA fragment (T-DNA) from its virulence Ti plasmid to the nuclear genome of the plant cells leading to genetically modified plant cells and plant-tumor formation. In plant tumor cells, the bacterial T-DNA encodes the production of opines including agrocinopine A used as nutrients and regulatory signals by agrobacteria which colonize the plant tumour tissues. Agrocinopine A is imported into the bacterial cytoplasm via the periplasmic binding protein AccA associated to a unique ABC-transporter (AccBCDE). Once in the cytoplasm, agrocinopine A is cleaved by the enzyme AccF into sucrose and L-arabinose-2-phosphate. However, Agrocin 84 produced by the non-pathogenic strain A. radiobacter K84 (which colonizes the same plant environment as the A. tumefaciens C58 pathogen) uses the same import system AccA and AccBCDE for penetrating into the cytoplasm of A. tumefaciens C58. AccF cleaves agrocin 84 into D-glucose-2-phosphate and the toxic moiety named TM84 which kills pathogen cells. In A. tumefaciens C58, agrocinopine A is proposed to interact with the transcriptional repressor AccR (dashed double lines), hence releasing acc and traR genes expression. Then, the transcriptional activator TraR interacts with quorum-sensing signals and promotes the expression of the tra, trb and rep genes which stimulate the biosynthesis of the quorum-sensing signals (traI), and amplification of copy number and conjugation of the Ti plasmid (regulation steps are indicated by double lines). In our work, we investigated the interactions between AccA and its ligands, as well as those between AccR and L-arabinose-2-phosphate and D-glucose-2-phosphate and their consequence on quorum-sensing and Ti plasmid transfer.
Fig 2.
(A) agrocinopine A and its derivatives. Reagents and conditions: (a) 1H-tetrazole, diisopropylamine, CH2Cl2, 2 h, 79% (b) 1H-tetrazole, CH2Cl2, 2 h, 64% (c) tBuOOH, octane, CH2Cl2, 2 h, 92%; (d) 60% aqueous acetic acid, 50°C, 30 min, 53% (e) H2, Pd/C,1 atm, 24 h, 83%(f) 1M methanolic MeONa, methanol, 30 min, 9+10 (g) K2CO3, methanol, 2h, 9, 34–68% (h) BnOH, 1H-tetrazole, CH2Cl2, 30 min, 84% (i) tBuOOH, octane, CH2Cl2, 30 min, 93%; (i) 60% aqueous acetic acid, 50°C, 30 min, 69% (k) H2, Pd/C, 1 atm, 24 h, quant. (l) isopropanol, 1H-tetrazole, CH3CN, 1 h, 50% (m) tBuOOH, octane, CH2Cl2, 30 min, quant. (n). 60% aqueous acetic acid, 50°C, 30 min, 64% (o) H2, Pd/C, 1 atm, 24 h, quant. (B) D-glucose-2-phosphate. Reagents and conditions: (a) BnOH, sulfamic acid, 80°C, neat, 10h, 22% (α/β = 5:2); (b) BnBr, NaH, DMF, rt, 18h, 86%; (c) TIBAL, toluene, 50°C, 60h, 26% (100% α); (d) 1H-tetrazole, (BnO)2-P-N(iPr)2, CH2Cl2, 2h, then m-CPBA, 0°C to rt, 2h, 84%; (e) H2, Pd/C, methanol, 18h, 87%.
Table 1.
Crystallographic data and refinement parameters.
Fig 3.
Ribbon representation of AccA in complex with agrocinopine A shown in its annealing Fo-Fc omit map contoured at 4δ.
Agrocinopine A is located in the cleft between the lobe 1 (residues 29–280 and 494–521) in slate and the lobe 2 (residues 285–489) in pink and is represented as yellow stick. The short hinge region is shown in red.
Fig 4.
Ligands and protein residues involved in the ligand binding are shown as stick. Hydrogen bonds are shown as dashed lines in black (for distances below 3.2 Å) and magenta (for distances between 3.2 and 3.4 Å). (A) agrocinopine A. (B) agrocin 84. (C) L-arabinose-2-phosphate. (D) D-glucose-2-phosphate. (E) Superimposition of the four ligands bound in the ligand binding site of AccA, agrocinopine A (yellow), agrocin 84 (orange), L-arabinose-2-phosphate (green) and D-glucose-2-phosphate (cyan) are shown as stick.
Fig 5.
Comparison of microcalorimetry derived enthalpy (ΔH, deep grey), entropic contribution (TΔS, grey) and free binding enthalpy (ΔG, light grey) at 293°K for agrocinopine A, agrocin 84, agrocinopine 3’-O-benzoate, L-arabinose-2-isopropylphosphate, L-arabinose-2-phosphate and D-glucose-2-phosphate.
Fig 6.
(A) AccR microcalorimetry measurements. The top panels show heat differences upon injection of ligand (L-arabinose-2-phosphate on the left and D-glucose-2-phosphate on the right) and lower panels show integrated heats of injection and the best fit (solid line) using Microcal Origin. Fitting values are indicated below. (B) Quantification of OC8HSL production and Ti plasmid conjugation induced by agrocinopine A, L-arabinose-2-phosphate and D-glucose-2-phosphate. Different A. tumefaciens C58 backgrounds were used as donor cells: C58 control, accF mutant, accF mutant complemented with p6000: accF wild type and accF mutant complemented with empty p6000). Results were obtained after 72 h of culture. SDs correspond to physical replicates. Experiments independently repeated between two and four times produce similar results.